| Background:Pancreatic cancer is a highly malignant tumor and its mortality rate ranks the fourth among malignant tumors.Due to its insidious onset and rapid progression,most of the patients had local invasion and distant metastasis at the first diagnosis,which has become the main factor for the high mortality rate of pancreatic cancer.Therefore,exploring the relevant molecular mechanisms of the progression of pancreatic cancer has guiding significance to understand the pathogenesis of pancreatic cancer and improve the diagnosis and treatment of pancreatic cancer.Long non-coding RNAs(LncRNAs)are a kind of polyadenylate RNAs with a length greater than 200 nt.It has tissue and disease specificity,and its sequence is highly conserved.In recent years,it has been found that there were a large number of dsregulated LncRNAs in pancreatic cancer.Although many tumor-associated LncRNAs have been reported,due to the extensive transcription of LncRNAs,there are still a large number of unannotated LncRNAs,and their role in tumors is unclear.Therefore,the exploration of tumor-related LncRNAs and its mechanism in tumor progression has become the spot in current pancreatic cancer research.In view of this,high-throughput gene chip technology and bioinformatics were conducted to detect and screen the gene expression in pancreatic cancer.We found that LncRNA RUNX1-IT1 was significantly overexpressed in pancreatic cancer tissues compared with normal pancreas tissues in the microarray.By retrieving the database(NCBI),we found RUNX1-IT1 was about 1502 bp,located in human chromosome 21.RUNX1-IT1 was reported in ovarian cancer in 2017,and it demonstrated that patients with high expression of RUNX1-IT1 had shorter disease-free survival and a higher risk of tumor recurrence.Later studies have found that RUNX1-IT1 was down-regulated in liver cancer and colorectal cancer,and it could inhibit tumor proliferation and migration,and induce apoptosis in vitro.At present,there are few studies on RUNX1-IT1 in tumors,and the deeper regulatory mechanism has not been reported.Moreover,the expression significance and mechanism of RUNX1-IT1 in pancreatic cancer are still unclear.Therefore,we intend to conduct a study of RUNX1-IT1 through clinical detection and analysis,functional experiments in vitro and in vivo,high-throughput sequencing of downstream targets and transcriptional regulation research,so as to clarify the role of RUNX1-IT1 in pancreatic cancer.RUNX1-IT1 is transcribed from the intron of the neighboring gene RUNX1,studies have shown that LncRNAs could exert a biological effect by regulating the expression of neighboring genes.RUNX1 is an important transcription factor in the blood system,and its abnormal expression is closely related to hematopoietic and myeloid leukemia.In recent years,it has been found that RUNX1 is also abnormally expressed in solid tumors and as a cancer-promoting or anticancer gene that affected the progression of malignant tumors.Therefore,we hypothesized that LncRNA RUNX1-IT1 might exert a biological effect in pancreatic cancer by acting on neighboring gene RUNX1.In summary,this study focused on whether RUNX1-IT1 could regulate pancreatic cancer proliferation,migration and invasion through RUNX1.The role of RUNX1-IT1 and its adjacent gene RUNX1 in the clinical prognosis of pancreatic cancer was analyzed.Furthermore,the high-throughput screening and the common target research of RUNX1-IT1 and RUNX1 are also the points.Finally,based on our clinical analysis and mechanism research,it is expected to clarify the molecular mechanism of RUNX-IT1 in promoting the progression of pancreatic cancer and provide new targets for the diagnosis and treatment of pancreatic cancer.Objectives:1.To determine the expression and significance of RUNX1-IT1 and RUNX1 in pancreatic cancer,and to evaluate whether RUNX1-IT1 and RUNX1 could be used as independent prognostic indicators for pancreatic cancer.2.To determine the molecular phenotype of RUNX1-IT1 and to determine whether RUNX1-IT1 exerts biological effects via RUNX1.To construct a model of orthotopic pancreatic tumor in nude mice,and to verify the effect of RUNX1-IT1/RUNX1 signaling axis on the invasion and metastasis of pancreatic cancer in vivo.3.To elucidate the downstream regulatory network and critical molecular mechanism of RUNX1-IT1 affecting pancreatic cancer progression.It mainly included two aspects: the mechanism of RUNX1-IT1 up-regulating RUNX1;the mechanism of RUNX1-IT1/RUNX1 acting on downstream target C-FOS.Methods:1.The expression of LncRNAs in pancreatic cancer was detected by Affymetrix HTA2.0Array.In combination with GEO database set(GSE15471,GSE16515),differentially expressed LncRNA RUNX1-IT1 in pancreatic cancer was screened and analyzed.Real-time fluorescence quantitative PCR(q PCR)and in situ hybridization were used to verify its expression level in clinical multi-center samples of pancreatic cancer.2.RUNX1-IT1 was divided into high and low expression groups according to in situ hybridization scores,and the differences in tumor size,TNM stage,pathological differentiation,clinical stage,and patient prognosis between the two groups were calculated.Univariate and multivariate analyses were used to assess whether RUNX1-IT1 could be an independent prognostic risk factor in patients with pancreatic cancer.3.To explore the biological function of RUNX1-IT1 at the cellular level.First,q PCR was used to detect the expression level of RUNX1-IT1 in pancreatic cancer cells,and cell lines with higher expression levels were selected for loss of function experiments.After transfected RUNX1-IT1 silencer in pancreatic cancer cells,we performed CCK8,Ed U,cell wound healing,transwell assays to observe the effects of RUNX1-IT1 on the proliferation,migration and invasion of pancreatic cancer cells,and to clarify the biological functions of RUNX1-IT1 in vitro.4.To detect RUNX1-IT1-mediated orthotopic liver metastasis in vivo.By inoculating the pancreas of nude mice with RUNX1-IT1 stable knockdown PANC-1 cells,the pancreatic orthotopic transplantation tumor model was constructed,and the small animal MRI and HE staining were used to determine the liver metastases and pathological characteristics.5.To clarify that RUNX1-IT1 perform biological functions through RUNX1.First,immunohistochemistry(IHC)was used to detect the expression of RUNX1 in pancreatic cancer tissues and analyze the relationship between its expression and clinical characteristics.Further statistical indicators such as correlation and subgroup analysis were used to clarify the expression relationship and prognostic significance of RUNX1-IT1 and RUNX1 in clinical samples of pancreatic cancer.Secondly,q PCR,Western blot,reporter gene,chromatin immunoprecipitation(CHIP)and other techniques were used to clarify the detailed mechanism of transcription regulation of RUNX1 by RUNX1-IT1.Finally,lentivirus stable cell lines with RUNX1-IT1 and RUNX1 were constructed to determine whether RUNX1-IT1 exerts biological effects through RUNX1 in vitro and in vivo.6.To investigated the common downstream target of RUNX1-IT1 and RUNX1.First,the RNA immunoprecipitation(RIP)experiment was used to determine whether the two interact to form a complex in pancreatic cancer;second,the downstream targets by RUNX1-IT1 and RUNX1 were screened and analyzed by RNA-seq and GSEA methods,and through q PCR,WB and cell function experiments to validate whether RUNX1-IT1 and RUNX1 could affect the proliferation,invasion and metastasis of pancreatic cancer via the C-FOS.Finally,the expression of RUNX1-IT1/RUNX1/C-FOS signal axis were detected in pancreatic cancer tissues.7.To clarify the detailed molecular mechanism of RUNX1-IT1-mediated RUNX1 regulating downstream target C-FOS.By constructing reporter genes and CHIP experiments,it was clear that RUNX1-IT1 activated the C-FOS transcription by interacting with RUNX1.Results:1.RUNX1-IT1 was highly expressed in pancreatic cancer tissues.By analyzing pancreatic cancer expression microarrays(GEO: GSE132956,GSE16515 and GSE15471,the top 2000 differential genes),we found that compared with normal pancreatic tissue,there were 40 up-regulated genes and 23 down-regulated genes in pancreatic cancer.In this study,we concentrated on 40 up-regulated genes because they had the greater potential to be used as early diagnostic markers or intervention targets.Through gene annotation,we found that RUNX1-IT1 was the only LncRNA in the up-regulated gene,while the others were coding genes.The coding potential of RUNX1-IT1 was further verified using an online coding assessment tool,which revealed that RUNX1-IT1 lacked protein coding capability(coding probability ability: 0.17;CP <0.364 is a non-coding sequence;http://lilab.research.bcm.edu/cpat/).Next,The q PCR analysis showed that RUNX1-IT1 was significantly up-regulated in pancreatic cancer tissues compared with adjacent tumor tissues(P <0.001).In situ hybridization was performed to verify RUNX1-IT1 expression in 175 pancreatic cancer samples and 13 normal pancreas samples from 3 clinical centers.Statistical analysis indicated that compared to normal pancreatic tissue,RUNX1-IT1 was significantly higher in multicenter pancreatic cancer samples(P <0.001).The results are consistent with our previous expression of RUNX1-IT1 in GEO datas.2.The elevated RUNX1-IT1 was closely related to the clinical staging of pancreatic cancer and was an independent risk factor for patient survival.We divided 175 samples into two groups with high and low expression according to the score of the ISH results.Statistical analysis showed that the expression of RUNX1-IT1 was positively correlated with tumor differentiation(P = 0.005),lymph node invasion(P = 0.001)and clinical stage(P = 0.007).Survival analysis revealed that the high expression of RUNX1-IT1 was closely related to the decrease in overall survival rate of pancreatic cancer(P <0.001).In addition,univariate analysis showed that poor differentiation(P = 0.015),lymph node metastasis(P = 0.002),high clinical stage(P = 0.002),and high expression of RUNX1-IT1(P <0.001)increased the risk of cancer-related death.Multivariate Cox regression analysis showed that RUNX1-IT1(P<0.001)was the independent prognostic factor.These data suggested that RUNX1-IT1 may play an important role in the progression of pancreatic cancer and had certain clinical value.3.Down-regulating the expression of RUNX1-IT1 inhibited the proliferation,invasion and metastasis of pancreatic cancer cells.CCK8 and Ed U assays showed that pancreatic cancer cell activity and proliferation rate were decreased in the RUNX1-IT1 knockdown group compared with the normal group;Cell wound healing and Transwell assays showed migration and invasion ability of pancreatic cancer cells were reduced in the RUNX1-IT1 knockdown group compared with the normal group.The orthotopic transplantation of pancreas in nude mice showed that the number of metastases on the liver surface of nude mice was significantly decreased after knockdown of RUNX1-IT1,indicated that the ability of pancreatic cancer cells to metastasize to the liver was reduced.The results suggested that RUNX1-IT1 affected the proliferation,invasion and migration of pancreatic cancer in vivo and in vitro.4.RUNX1 was highly expressed and played a pro-oncogenic role in pancreatic cancer.First,immunohistochemical experiments showed that RUNX1 was highly expressed in pancreatic cancer,and was closely related to clinical stage,tumor size,lymphatic metastasis and other indicators of clinical patients(P <0.05).Multivariate analysis found that RUNX1 was an independent prognostic factor for pancreatic cancer patients(P = 0.001).Cell function experiments showed that after knockdown of RUNX1,the proliferation,invasion and migration ability of pancreatic cancer cells were significantly decreased.The pancreatic orthotopic transplantation tumor model showed that in the group that knockdown of RUNX1,the number of micrometastasis on the liver surface of nude mice was decreased,indicated the orthotopic liver metastasis ability of pancreatic cancer cells was reduced.These datas suggested that RUNX1 was highly expressed in pancreatic cancer tissues,and may be involved in regulating the proliferation,invasion and migration of pancreatic cancer cells.5.RUNX1-IT1 played a biological function via positively regulating the expression of RUNX1.Correlation analysis showed that there was a positive correlation between RUNX1-IT1 and RUNX1 m RNA expression in pancreatic cancer tissues.The subgroup Kaplan-Meier analysis was performed based on RUNX1-IT1 ISH and RUNX1 IHC scores.The results showed that the group with high expression of RUNX1-IT1 and RUNX1 had the worst prognosis(P<0.001).Secondly,downregulation of RUNX1-IT1 resulted in reduced m RNA and protein levels of RUNX1,suggesting a positive regulatory relationship between the RUNX1-IT1 and RUNX1.By constructing the RUNX1 promoter reporter gene and co-transfecting with the overexpression or interference vector of RUNX1-IT1,the fluorescence intensity of the RUNX1 promoter was up-regulated or down-regulated,suggesting that RUNX1-IT1 could regulate the RUNX1 promoter at the transcriptional level.Ch IP-PCR analysis showed that the promoter region of RUNX1 was modified by H3K27 Ac.Further experiments showed that the expression alteration of RUNX1-IT1 could affect the H3K27 Ac level of the promoter region of RUNX1.In vitro recovery experiments showed that overexpression of RUNX1-IT1 promoted the proliferation and invasion and migration of pancreatic cancer,and down-regulated expression of RUNX1 attenuated the cancer-promoting effect of RUNX1-IT1.The results of orthotopic pancreas transplantation tumor also suggested that down-regulation of RUNX1 expression could attenuate RUNX1-IT1-induced orthotopic liver metastasis.In brief,these results indicated that RUNX1-IT1 may exert a cancer-promoting biological effect through RUNX1.6.To elaborate that C-FOS was the common downstream target of RUNX1-IT1 and RUNX1.Based on the consistency of RUNX1-IT1 and RUNX1 expression in pancreatic cancer,we intended to investigate whether they were jointly participated in downstream gene regulation.First,the RIP experiment found that RUNX1-IT1 and RUNX1 could bind to each other in tumor cells,suggesting that they may play a common molecular regulatory function in pancreatic cancer.After downregulation of RUNX1-IT1 or RUNX1,RNA sequencing was performed to screen downstream targets respectively.The KEGG and GSEA analysis suggested that both RUNX1-IT1 and RUNX1 were involved in the regulation of TNF signaling pathways,and further differential gene screening suggested that C-FOS gene was enriched in TNF signaling pathway and was correlated with RUNX1-IT1 and RUNX1.The q PCR and western blot assays identified that RUNX1-IT1 and RUNX1 could regulate C-FOS gene expression.Then,the cell function recovery experiment showed that downregulating C-FOS expression could weaken the ability of promoting cell proliferation,migration and invasion of RUNX1-IT1 and RUNX1.To further evaluate the relationship between RUNX1-IT1 and RUNX1 expression and C-FOS signaling pathway in clinical tissues,IHC assay was performed.The results showed that RUNX1-IT1,RUNX1,C-FOS and its downstream molecules such as MMP9 and CCND1 were significantly correlated in multicenter samples of pancreatic cancer.These results suggested that RUNX1-IT1 and RUNX1 may interact and participate in the regulation of downstream target gene C-FOS,thus further affecting biological processes such as proliferation,invasion and migration of pancreatic cancer.7.RUNX1-IT1 activated C-FOS gene transcription by interacting with RUNX1.First,the binding sites of RUNX1 in C-FOS promoter were predicted through bioinformatics,and it was found that RUNX1 may bind to C-FOS promoter via four binding sites.Then C-FOS promoter wild-type and mutant vectors were constructed and reporter gene detection was carried out.We found that C-FOS promoter luciferase activity was significantly reduced in the RUNX1 knockdown group compared to the control group,while the pattern was reversed in the RUNX1 overexpressed group.The RUNX1-IT1 downregulated and overexpressed groups also had similar results.Meanwhile,the reporter gene of the mutant vector and Ch IP-PCR analysis showed RUNX1 could obviously affect the transcriptional activity of C-FOS promoter-related three sites.In addition,luciferase reporter gene assay was performed again to verify whether RUNX1-IT1 was required for RUNX1 binding to C-FOS promoter.In the overexpressing RUNX1 pancreatic cancer cells,down-regulation of RUNX1-IT1 significantly reduced C-FOS promoter activity,suggesting that knockdown of RUNX1-IT1 attenuated RUNX1-mediated regulation on C-FOS promoter activity.Further Ch IP-PCR analysis showed that knockdown of RUNX1-IT1 reduced the enrichment of RUNX1 in three loci of C-FOS promoter.These results suggested that RUNX1-IT1 regulated C-FOS gene transcription by affecting RUNX1 binding to C-FOS promoter.Furthermore,this observation led us to investigate whether RUNX1-IT1 could act as an independent regulatory RNA that binds to the C-FOS promoter site.Using Ch IRP,a technique for detecting RNA-DNA interactions,we isolated fragments of chromatin that bound of RUNX1-IT1.Ch IRP-PCR analysis showed that RUNX1-IT1 had a low occupation rate in the C-FOS promoter region,indicating that RUNX1-IT1 could not directly bind to the promoter of the C-FOS gene,and it further revealed that RUNX1-IT1 mediated C-FOS transcriptional regulation was required through interaction with RUNX1.These findings confirmed that RUNX1-IT1 may be a transcriptional coordinating factor for RUNX1 mediated C-FOS regualtion.Conclusions:RUNX1-IT1 was as a pro-oncogene involved in the proliferation,invasion and metastasis in pancreatic cancer.Clinical analysis showed that RUNX1-IT1 and RUNX1 could be used as independent risk factors for assessing the prognosis of pancreatic cancer.In terms of molecular mechanism,RUNX1-IT1 influences the progression of pancreatic cancer by regulating the expression of RUNX1 and promoting the regulation of RUNX1 on the C-FOS gene promoter,thus clarifying a new molecular regulatory mechanism for the progression of pancreatic cancer.Finally,the newly discovered RUNX1-IT1 /RUNX1/C-FOS signaling axis may provide theoretical basis and potential therapeutic targets in pancreatic cancer. |
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